Project Summary The plant hormone auxin is involved in virtually all aspects of plant growth and development. Over the years we and others have demonstrated that auxin acts by stimulating the degradation of a family of transcriptional repressors called the Aux/IAA proteins, through the action of the ubiquitin protein ligase SCFTIR1/AFB. Although this basic pathway is well established, we still know very little about how auxin regulates plant growth and development. Strikingly, auxin-regulated genes differ dramatically between cell types and organs, consistent with the diverse roles of the hormone. The basis for this specificity is unknown. Similarly, the activities of the Aux/IAA and ARF proteins are poorly characterized and the transcriptional networks (GRNs) that mediate various auxin-regulated growth processes have not been defined. Finally, we know little about how auxin signaling is integrated with other environmental and genetic signaling pathways. During the last grant period we demonstrated that the 6 members of the AFB family of F-box proteins act in an additive fashion but may also have specific functions. In addition, our recent ChIPseq experiments indicate that the AFB proteins are bound to chromatin adjacent to auxin-regulated genes and are recruited to these sites by auxin. We speculate that the close association of SCFAFB with chromatin permits rapid de- repression of auxin responsive genes. We also made significant advances in our understanding of the Aux/IAA proteins and the architecture of the auxin signaling network. Our work shows that the Aux/IAA genes function as regulatory nodes that integrate environmental signals with the auxin gene regulatory network. Most exciting, we have shown that the DREB2-dependent but ABA-independent abiotic stress response pathway acts through the Aux/IAAs to mediate drought tolerance. Further we demonstrate that this pathway regulates the levels of secondary products called glucosinolates and that these compounds promote stomatal closure and drought tolerance. These studies indicate that glucosinolates have previously unknown signaling properties. The long-term goals of this proposal are to determine the molecular basis of auxin signaling and to characterize the auxin-based regulatory networks that control plant growth and development. Our specific aims are to; 1) Investigate the role of the IAA5,6,19 protein in DREB2-mediated drought tolerance, 2) Determine the mechanism of Aux/IAA-based transcriptional repression, 3) Explore the specificity of the AFB and Aux/IAA proteins, and 4) Investigate recruitment of AFBs to chromatin. These studies address a number of key issues in cellular regulation and will have important implications for human health. The ubiquitin pathway and the SCFs in particular are involved in diverse disease processes including numerous cancers. Because SCFTIR/AFB1 is one of the best-characterized E3 complexes in any organism, this work provides a unique opportunity to advance our understanding of this critical aspect of human disease.